Screening of Clostridium sp. for butanol production from Sato (Thai rice wine) factory wastewater

Abstract

The production of Sato, a Thai traditional beverage, yields Sato wastewater (STW), which contains residual rice constituents with high chemical and biological oxygen demands and so forms an environmental problem. In addition, STW often smells rotten from the formation of hydrogen sulfide. To solve this problem, the composition of STW was analyzed for its physiological and chemical characteristics to evaluate its potential as a substrate for ABE fermentation using Clostridium spp., which has never been reported for use in acetone-butanol-ethanol (ABE) fermentation before. Solvent-producing Clostridium isolates were obtained from environmental sources in Thailand, and that producing the highest butanol level (isolate CUEA02 from the sludge of a biodiesel plant) was selected. The butanol production was 8.32 ± 0.08 g/L in flask fermentation under an initial pH of 6.5, 35 ˚C, 10% (v/v) inoculum size, and 50 g/L of glucose. Whole-genome sequencing analysis was used to identify this strain and its ability to use different carbon sources for ABE fermentation was evaluated. Isolate CUEA02 was closely related to Clostridium beijerinckii with an average nucleotide identity of 95.14% and could grow and produce butanol in various carbon sources, especially starch and carboxymethyl cellulose. The STW was diluted 1:10, 1:20, 1:30, and 1:40 for ABE production by CUEA02, where the obtained butanol yield (0.23 g/g) under an anaerobic condition was highest with the 1:10 diluted STW. To improve the butanol production, the initial pH was adjusted from 4.75 to 6.5, resulting in a 2.13-fold enhanced butanol yield (0.49 g/g). Furthermore, adding yeast extract at 2 g/L enhanced butanol production in STW utilizing C. beijerinckii CUEA02 by up to 29%.